Rhizomucor miehei aspartic protease is a microbial protease suitable for milk-clotting purposes. Preparations of milk-clotting enzymes are used in the food industry, for instance in cheese manufacturing.
Rhizomucor miehei aspartic protease is the most commonly used milk-clotting enzyme, due to its low cost and favorable performance (Sternberg, M. Z. (1971) “Crystalline milk-clotting protease from Mucor miehei, and some of its properties” J. Dairy Sci., Vol. 54, pp 159-167). This protease can also be produced heterologously via recombinant DNA technology in filamentous fungi (Boel, E. et al. European Patent EP 238 023).
A problem with the production of proteases via microbial fermentation is the expression of other undesirable enzymes. Due to an increasing demand in the market for pure enzyme preparations, several methods have been developed in the art for either the separation and purification of desirable enzymes from enzyme mixtures or for the selective inactivation of undesirable enzymes.
U.S. Pat. No. 2,683,682 (1954) discloses the differential inactivation of proteases or amylases from mixtures of both enzyme types. The method comprises adjusting the pH of the aqueous solution between 3.0 and 4.5 (to selectively inactivate amylase) or between 7.0 and 10.5 (to selectively inactivate protease). The method also comprises maintaining the mixture at a temperature comprised between about 5° C. and 60° C. for a period of time sufficient to inactivate the undesired enzyme, generally for about 0.5 hours or less for higher temperatures and 20 hours for lower temperatures. Treatment of enzyme mixtures derived from malted wheat flour or malted barley at pH 3.6 and 50° C. for 0.5 hours leads to a protease recovery of up to 66% and amylase deactivation of 99.7%. At the same pH but 5° C. for 20 hours the protease recovery is 85%, with an amylase deactivation of 99.6%.
U.S. Pat. No. 4,086,139 (1978) describes the selective inactivation of amylase in enzyme mixtures comprising protease and amylase. The inactivation of amylase occurs by treating the enzyme mixture with an oxidizing agent selected from chlorite and hypochlorite ions. The ions are added to the enzyme mixture in sufficient amount to inactivate the amylase and leave the protease intact, so that further purification steps are avoided. The temperature and pH of the treatment are not critical as far as they are not detrimental to the protease. This method can treat enzyme compositions derived from animal organs (e.g. crude animal organ extracts) or bacteria like Bacillus subtilis or licheniformis (e.g. fermentation broths). With this method, more than 80% of the amylase is inactivated leaving more than 80% of the protease activity intact.
International Patent Application WO 97/20921 (published Jun. 12, 1997) describes a method for the selective inactivation of at least one undesirable enzyme from an enzyme mixture comprising desirable and undesirable enzymes. The enzyme mixture is treated at a pH lower than 5 and at a temperature from 2 to 75° C. for at least 20 seconds, and/or at a pH higher than 9 and at a temperature from 2 to 75° C. for at least 20 seconds. According to this method, amylase is completely inactivated by treatment of an enzyme mixture comprising amylase and cellulase at pH 3.5 for 1 min at 70° C., while 96% of the cellulase activity remains intact. On the other hand, only 2% of the protease activity is left when enzyme mixture comprising lipase and protease is treated at pH 3.5, 45° C. for 60 minutes. In mixtures comprising cellulase and protease (Example 7), the protease is completely inactivated by treating at 3° C. or 25° C. for 60 minutes and at a pH of 2.5.
U.S. Pat. No. 5,139,943 (1992) describes a method for selective recovery of microbial produced chymosin from mixtures of polypeptides or enzymes produced by fermentation, for example α-amylase. This method is based on the use of a two phase, liquid-liquid system having a partition coefficient for chymosin greater than about 85. The two-phase system is obtained by adding PEG and a salt, for example a phosphate or a sulphate, to the aqueous enzyme composition. The chymosin is selectively extracted into the organic phase while the amylase stays in the aqueous phase. Chymosin can be recovered from the PEG via ion exchange chromatography. By using a pH lower than 3, partition coefficients for chymosin can be obtained of about 1000, allowing full separation from amylase and a chymosin recovery of about 96-98%.
For chymosin obtained by recombinant techniques, U.S. Pat. No. 4,743,551 (Subramanian) and U.S. Pat. No. 4,721,673 (Subramanian et al.) propose dye affinity ligand adsorption and U.S. Pat. No. 5,122,467 (Heinsohn et al.) proposes adsorption to phenyl SEPHAROSE® matrix (SEPHAROSE® matrix is the trade mark of GE Healthcare Biosciences; the corresponding products are based on agarose). U.S. Pat. No. 5,122,467 states that a comparison was made between phenyl SEPHAROSE® matrix and agaroses with other functionalities (including octyl), and it was concluded that only the former provides the required selectivity for chymosin in fermentation broths. Experiments have also been presented to use other ligands containing aromatic rings in WO 96/00735 (Burton et al.) and WO 96/09116 (Burton et al.).
In a recent study by Burton et al. (“One-step purification of chymosin by mixed mode chromatography” in Biotech. Boeing. 56(1) (1997) 4555) a number of chargeable and non-chargeable aromatic ligands have been examined for adsorption of chymosin from a fermentation broth.
Purification of microbial protease can be achieved by binding of microbial protease to an appropriate chromatographic resin. Suitable resins are for instance ion exchange resins. Ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to an immobilized ion exchange group of opposite charge. Separation is obtained because different substances have different degrees of interaction with the ion exchanger due to differences in their charges and charge densities. Molecules are bound to ion exchangers when they carry a net charge opposite to that of the ion exchanger. The binding is electrostatic and reversible. Ion exchange chromatography is a technique which offers different selectivities using either anion or cation exchangers. Separation can be obtained by differences in charge of the biological compounds. Changing the pH alters the charge characteristics of the sample components and can thus modify the separation.
Anion exchangers, for instance Q-SEPHAROSE® matrix, can be used if a microbial protease solution at a pH above its isoelectric point (IP) is applied to an anion exchanger equilibrated at the same pH. The bound microbial protease can be eluted (desorbed), free of contaminating proteins by increasing the ionic strength and/or changing the pH. The change in ionic strength and/or pH during desorption can take place as a stepwise or continuous gradient.
The same separation can be achieved with a cation exchanger, for instance SP-SEPHAROSE® matrix, if a microbial protease preparation is applied to the resin below its IP.
Other suitable resins are hydrophobic interaction media. Using a hydrophobic interaction medium, separation can be based on the differences in hydrophobicity. Different hydrophobic interaction resins are available containing different ligands for instance ethyl, propyl, butyl, phenyl, and octyl. By applying an aqueous microbial protease solution under conditions permitting binding of microbial protease to the resin, it is possible to separate microbial protease from contamination compounds
Some purification methods described in the art have the disadvantage of being laborious. In general, said methods are characterized by a low selectivity towards protease and/or by a relatively high loss in protease activity and/or by excessive use of salts and/or solvents. Despite the number of previously suggested purification protocols, there is still a need for improvements relating to yield/recovery, purity, specific microbial protease activity, simplicity of operation, need for elution agents and salt burden amongst others.